PROJECT SUMMARY Interleukin-37 is a remarkable IL-1 family member that can decrease inflammation and block cancer progression. The discovery over 30 years ago of interleukin-1? (IL-1?) marked a revolution in biology with the identification of a single highly potent protein that induces inflammation at picamolar concentrations, yet IL-37 presents a similar revolution whereby IL-37 can reverse these effects. However, there have been no studies aimed at elucidating the molecular details of IL-37 interactions, despite the high potential of exploiting such a pathway therapeutically, either through agonists or through recombinant IL-37 itself. Thus, it is our goal here to begin elucidating the molecular interactions that underlie the anti-inflammatory activity of IL-37. The novelty of IL-37 is that it dimerizes unlike any other IL-1 family member, which likely occludes an active binding surface and may be the underlying reason as to why its receptor interactions have been difficult to characterize. We have made significant strides towards identifying the IL-37 homodimer surface in an effort to engineer an active monomer and we have cloned and begun characterizing multiple receptors predicted to engage IL-37. We hypothesize that the IL-37 monomer defines the active IL-37 form capable of binding a competent signaling complex that the dimer inhibits. We will test this hypothesis through the following aims: Aim 1) Determine the molecular basis of IL-37 dimerization and its role in IL-37 activity. Capitalizing on our preliminary NMR studies, we will utilize specific labeling techniques together with NMR to determine how IL-37 dimerizes. Established methods will be utilized to disrupt this interface in order to produce an IL-37 monomer as others and we have previously described in order to compare the activities of the IL-37 dimer with that of an engineered IL-37 monomer. Aim 2) Identify the direct targets of IL-37 and the sequence of binding events. Both unbiased as well as directed methods will be used to determine the cellular targets of IL-37 and identify the binding events. Established cross-linking/mass spectrometry methods will be used to identify the direct targets of IL-37 while we have simultaneously begun producing recombinant proteins previously proposed to engage IL-37 (i.e., IL- 18R? and IL1-R8).